This is a first time revised application intended to assess biochemical regulation of sperm motility. The long-term goal is to define the biochemical mechanisms underlying development and regulation of motility with emphasis on considerations of phosphorylation of proteins associated with the axoneme. Levels of phosphorylation are a balance between the action of protein kinases and protein phosphatases. The applicant has identified a sperm-specific protein phosphatase, PP1g2 that is involved in regulation of sperm motility. High activity of this enzyme results in low sperm motility, whereas vigorously motile sperm exhibit low enzyme activity. Specific inhibition of PP1g2 leads to initiation of motility in immotile sperm and to the stimulation of velocity and flagellar beat amplitude in motile sperm. Genetic and biochemical studies support the hypothesis of the applicant that PP1g2 is a key component regulating sperm phosphorylation and, hence, motility. The focus of this particular proposal is to study the biochemical mechanisms regulating PP1g2. The investigator hypothesizes that two sperm-specific regulatory proteins, P33 and P72, control the activity and the sub cellular localization of PP1g2. Furthermore, he believes that the interaction of PP1g2 with P33 is regulated by glycogen synthase kinase-3 phosphorylation. Three Specific Aims to test these ideas are formulated as follows: (1) To purify and sequence the inhibitor P33 and to determine how it regulates PP1g2. (2) To determine the molecular basis for the sub cellular targeting of PP1g2 to the sperm flagellum and the anterior region of the sperm head. (3) To determine how PP1g2 and its activating enzyme glycogen synthase kinase-3 are regulated by sperm intracellular second messengers. Elucidation of such novel sperm biochemical mechanisms should lead to a better understanding of male gamete function, result in novel approaches for the regulation of sperm motility, and could enable better diagnosis and treatment of male factor infertility.